In multicellular organisms, homeostasis is maintained by balancing the rate of cell proliferation against the rate of cell death. Cell proliferation is influenced by numerous growth factors and the expression of proto-oncogenes, which typically encourage progression through the cell cycle. In contrast, numerous events, including the expression of tumor suppressor genes, can lead to an arrest of cellular proliferation.
In differentiated cells, a particular type of cell death called apoptosis occurs when an internal suicide program is activated. This program can be initiated by a variety of external signals as well as signals that are generated within the cell in response to, for example, genetic damage. For many years, the magnitude of apoptotic cell death was not appreciated because the dying cells are quickly eliminated by phagocytes, without an inflammatory response.
The mechanisms that mediate apoptosis have been intensively studied. These mechanisms involve the activation of endogenous proteases, loss of mitochondrial function, and structural changes such as disruption of the cytoskeleton, cell shrinkage, membrane blebbing, and nuclear condensation due to degradation of DNA. The various signals that trigger apoptosis are thought to bring about these events by converging on a common cell death pathway that is regulated by the expression of genes that are highly conserved from worms, such as C. elegans, to humans. In fact, invertebrate model systems have been invaluable tools in identifying and characterizing the genes that control apoptosis. Through the study of invertebrates and more evolved animals, numerous genes that are associated with cell death have been identified, but the way in which their products interact to execute the apoptotic program is poorly understood.
Caspases, a class of proteins central to the apoptotic program, are responsible for the degradation of cellular proteins that leads to the morphological changes seen in cells undergoing apoptosis. Caspases (cysteinyl aspartate-specific proteinases) are cysteine proteases having specificity for aspartate at the substrate cleavage site. Generally, caspases are classified as either initiator caspases or effector caspases, both of which are zymogens that are activated by proteolysis that generates an active species. An effector caspase is activated by an initiator caspase which cleaves the effector caspase. Initiator caspases are activated by an autoproteolytic mechanism that is often dependent upon oligomerization directed by association of the caspase with an adapter molecule.
Many caspases and proteins that interact with caspases possess domains of about 60 amino acids called a caspase recruitment domain (CARD). Hofmann et al. (TIBS 22:155, 1997) and others have postulated that certain apoptotic proteins bind to each other via their CARDs and that different subtypes of CARDs may confer binding specificity, regulating the activity of various caspases, for example. The functional significance of CARDs have been repeatedly demonstrated. For example, Duan et al. (Nature 385:86, 1997) showed that deleting the CARD at the N-terminus of RAIDD abolished the ability of RAIDD to bind to caspases.
Caspase-1 is an example of an initiator caspase. Caspase-1 was first discovered as the protease responsible for the conversion of the inactive precursor of IL-1 to the mature proinflammatory cytokine (caspase-1 was originally termed interleukin-1 converting enzyme, ICE). Caspase-1 also processes the inactive precursor of the cytokine IL-18 into an active form. Caspase-1 is synthesized as a single chain zymogen consisting of an N-terminal CARD containing prodomain and a large (p20) and small (p 10) catalytic domain. Caspase-1 is thought to oligomerize upon the receipt of a proinflammatory signal and autoprocess to generate an active heterodimeric protease consisting of the p20 and p10 subunits.
RIP2 (CARDIAK/RICK) binds caspase-1 via an interaction between the CARD domain of RIP2 and the CARD domain of caspase-1. This interaction results in the processing and activation of caspase-1. Thus, RIP2 is thought to be an upstream activator adaptor of caspase-1. Conversely, the activation of caspase-1 and subsequent generation of IL-1 P is regulated by a CARD domain-containing decoy molecule termed ICEBERG. This decoy attenuates inflammation by binding to the CARD domain of caspase-1 and inhibiting or displacing the upstream activator RIP2. ICEBERG is induced by proinflammatory stimuli and thus appears to be part of a negative feedback loop that shuts off IL-1xcex2 generation and thus dampens the inflammatory response (Humke et al., Cell 103:99, 2000).
In addition to its role in inflammation via IL-1xcex2 processing, caspase-1 also appears to participate in cell death pathways. For example, overexpression of caspase-1 in Rat-1 fibroblasts induces apoptosis that can be suppressed by overexpression of antiapoptotic genes such as Bcl-2 (Miura et al., Cell 75:653, 1993).
Caspase-9 activation may precede the activation of all other cell death-related caspases in the mitochondrial pathways of apoptosis (Slee et al., J. Cell Biol. 144:281-292, 1999). Inactive procaspase-9 is activated by interaction with a complex which includes Apaf-1, a CARD-containing protein, and other factors (Li et al., Cell 91:479, 1997; Srinivasula et al., Mol. Cell 1:949-959, 1998). Recognition of procaspase-9 by Apaf-1 occurs primarily through the interaction of the CARD of Apaf-1 with the prodomain of caspase-9. The CARD of Apaf-1 shares about 20% sequence identity with the prodomain of procaspase-9. The prodomain of caspase-9 is a member of the CARD family of apoptotic signaling motifs (Hofmann and Bucher, Trends in Biochem. Sci. 22:155-156, 1997). A similar domain is present in caspase activating proteins CED-4 and RAIDD/CRADD as well as in initiator caspases CED-3 and caspase-2/ICH-1 (Duan and Dixit, Nature 385:86-89, 1997; Ahmad et al., Cancer Res. 57:615-619, 1997; Alnemri et al., Cell 87:171, 1996). Apaf-1 can bind several other caspases, e.g., caspase-4 and caspase-8 (Inohara et al., J. Biol. Chem. 273:12296-12300, 1998).
Nuclear factor-xcexaB (NF-xcexaB) is a transcription factor expressed in many cell types and which activates homologous or heterologous genes that have xcexaB sites in their promoters. Molecules that regulate NF-xcexaB activation play a critical role in both apoptosis and inflammation. Quiescent NF-xcexaB resides in the cytoplasm as a heterodimer of proteins referred to as p50 and p65 and is complexed with the regulatory protein IxcexaB. NF-xcexaB binding to IxcexaB causes NF-xcexaB to remain in the cytoplasm. At least two dozen stimuli that activate NF-xcexaB are known (New England Journal of Medicine 336:1066, 1997) and they include cytokines, protein kinase C activators, oxidants, viruses, and immune system stimuli. NF-xcexaB activating stimuli activate specific IxcexaB kinases that phosphorylate IxcexaB leading to its degradation. Once liberated from IxcexaB, NF-xcexaB translocates to the nucleus and activates genes with xcexaB sites in their promoters. The proinflammatory cytokines TNF-xcex1 and IL-1 induce NF-xcexaB activation by binding their cell-surface receptors and activating the NF-xcexaB-inducing kinase, NIK, and NF-xcexaB. NIK phosphorylates the IxcexaB kinases xcex1 and xcex2 which phosphorylate IxcexaB, leading to its degradation.
NF-xcexaB and the NF-xcexaB pathway has been implicated in mediating chronic inflammation in inflammatory diseases such as asthma, ulcerative colitis, rheumatoid arthritis (Epstein, New England Journal of Medicine 336:1066, 1997) and inhibiting NF-xcexaB or NF-xcexaB pathways may be an effective way of treating these diseases. NF-xcexaB and the NF-xcexaB pathway has also been implicated in atherosclerosis (Navab et al., American Journal of Cardiology 76:18C, 1995), especially in mediating fatty streak formation, and inhibiting NF-xcexaB or NF-xcexaB pathways may be an effective therapy for atherosclerosis. Among the genes activated by NF-xcexaB are cIAP-1, cIAP-2, TRAF1, and TRAF2, all of which have been shown to protect cells from TNF-xcex1 induced cell death (Wang et al., Science 281:1680-83, 1998). CLAP, a protein which includes a CARD, activates the Apaf-1-caspase-9 pathway and activates NF-xcexaB by acting upstream of NIK and IxcexaB kinase (Srinivasula et al., supra).
Bcl-2 family proteins are important regulators of pathways involved in apoptosis and can act to inhibit or promote cell death. Expression of certain anti-apoptotic Bcl-2 family members is commonly altered in cancerous cells, suppressing programmed cell death and extending tumor growth. Among the anti-apoptotic Bcl-2 family members thus far identified are Boo, Bcl-2, Bcl-xL, Bcl-w, NR-13, A1, and Mcl-2. Pro-apoptotic Bcl-2 family members include Bax, Bak, Bad, Bik, Bid, Hrk, Bim, and Bok/Mtd. Significantly, the anti-apoptotic Bcl-2 family member, Bcl-xL, has been shown to interact with Apaf-1 and block Apaf-1-dependent caspase-9 activation (Hu et al., Proc. Nat""l. Acad. Sci. 95:4386-4391, 1998). Boo, another anti-apoptotic Bcl-2 family member, interacts with Apaf-1 and caspase-9. Bak and Bik, pro-apoptotic Bcl-2 family members, can disrupt the association of Boo with Apaf-1 (Song et al., EMBO J. 18:167-178, 1999). Boo is thought to be involved in the control of ovarian atresia and sperm maturation. Diva, another member of the Bcl-2 family, inhibits binding of Bc1-xL to Apf-1, preventing Bcl-xL from binding to Apaf-1.
Neurotrophins (e.g., NGF), which are best known as neuronal survival factors, can mediate apoptosis via the p75 neurotrophin receptor (p75NTR). It is thought that p75NTR activation can lead to NF-xcexaB activation (Carter et al., Science 272:542-545, 1996). It has been proposed that p75NTR-mediated cell death acts to ensure rapid cell death when a neuron is unable to obtain sufficient neurotropins. This mechanism could, for example, cause the elimination of neurons that reach an inappropriate target or that reach an appropriate target at an inappropriate time (Miller and Kaplan, Cell Death and Diff. 5:343-345, 1998).
The present invention is based, at least in part, on the discovery of genes encoding CARD-5. Full-length cDNAs encoding murine and human CARD-5 are presented.
CARD-5 is an intracellular protein that is predicted to be involved in regulating caspase activation. CARD-5 is found to activate the NF-xcexaB pathway and to bind to the CARD domains of caspase-1, CARD-7, and CARD-5 itself.
The 777 nucleotide murine CARD-5 cDNA described below (SEQ ID NO:1) has a 579 nucleotide open reading frame (nucleotides 89 to 667 of SEQ ID NO: 1; SEQ ID NO:3) which encodes a 193 amino acid protein (SEQ ID NO:2). Murine CARD-5 contains a CARD domain which extends from amino acid 110 to amino acid 193 of SEQ ID NO:2 (SEQ ID NO:7).
The 740 nucleotide human CARD-5 cDNA described below (SEQ ID NO:4) has a 585 nucleotide open reading frame (nucleotides 54 to 638 of SEQ ID NO:4; SEQ ID NO:6) which encodes a 195 amino acid protein (SEQ ID NO:5). Human CARD-5 contains a CARD domain which extends from amino acid 111 to amino acid 195 of SEQ ID NO:5 (SEQ ID NO:8).
Like other proteins containing a CARD domain CARD-5 participates in the network of interactions that lead to caspase activity. Human CARD-5 likely plays functional roles in caspase activation similar to that of FADD. CARD-5 through its CARD and pyrin domains may interact with other proteins having a CARD domain and/or a pyrin domain. For example, CARD-5 may serve as bridge to link a CARD domain-containing protein and/or a pyrin domain containing protein thus directly or indirectly activating signaling pathways, e.g., cell signaling pathways. CARD-5 may bind to and activate a CARD-containing caspase via a CARDxe2x80x94CARD interaction, leading to apoptotic death of the cell and/or cytokine processing that leads to inflammation. CARD-5 molecules are useful as modulating agents in regulating a variety of cellular processes including cell growth and cell death. In one aspect, this invention provides isolated nucleic acid molecules encoding CARD-5 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of CARD-5 encoding nucleic acids.
The invention encompasses methods of diagnosing and treating patients who are suffering from a disorder associated with an abnormal level or rate (undesirably high or undesirably low) of apoptotic cell death, abnormal activity of the Fas/APO-1 receptor complex, abnormal activity of the TNF receptor complex, abnormal inflammatory response, or abnormal activity of a caspase by administering a compound that modulates the expression of CARD-5 (at the DNA, mRNA or protein level, e.g., by altering mRNA splicing) or by altering the activity of CARD-5. Examples of such compounds include small molecules, antisense nucleic acid molecules, ribozymes, and polypeptides.
Certain disorders are associated with an increased number of surviving cells, which are produced and continue to survive or proliferate when apoptosis is inhibited or occurs at an undesirably low rate. Compounds that modulate the expression or activity of CARD-5 can be used to treat or diagnose such disorders. These disorders include cancer (particularly follicular lymphomas, chronic myelogenous leukemia, melanoma, colon cancer, lung carcinoma, carcinomas associated with mutations in p53, and hormone-dependent tumors such as breast cancer, prostate cancer, and ovarian cancer). Such compounds can also be used to treat viral infections (such as those caused by herpesviruses, poxyiruses, and adenoviruses). Failure to remove autoimmune cells that arise during development or that develop as a result of somatic mutation during an immune response can result in autoimmune disease. Thus, autoimmune disorders can be caused by undesirably low levels of apoptosis. Accordingly, modulators of CARD-5 or activity or expression can be used to treat autoimmune disorders (e.g., systemic lupus erythematosis, immune-mediated glomerulonephritis, and arthritis).
Many diseases are associated with an undesirably high rate of apoptosis. Modulators of CARD-5 expression or activity can be used to treat or diagnose such disorders. For example, populations of cells are often depleted in the event of viral infection, with perhaps the most dramatic example being the cell depletion caused by the human immunodeficiency virus (HIV). Surprisingly, most T cells that die during HIV infections do not appear to be infected with HIV. Although a number of explanations have been proposed, recent evidence suggests that stimulation of the CD4 receptor results in the enhanced susceptibility of uninfected T cells to undergo apoptosis. A wide variety of neurological diseases are characterized by the gradual loss of specific sets of neurons. Such disorders include Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa, spinal muscular atrophy, and various forms of cerebellar degeneration. The cell loss in these diseases does not induce an inflammatory response, and apoptosis appears to be the mechanism of cell death. In addition, a number of hematologic diseases are associated with a decreased production of blood cells. These disorders include anemia associated with chronic disease, aplastic anemia, chronic neutropenia, and the myelodysplastic syndromes. Disorders of blood cell production, such as myelodysplastic syndrome and some forms of aplastic anemia, are associated with increased apoptotic cell death within the bone marrow. These disorders could result from the activation of genes that promote apoptosis, acquired deficiencies in stromal cells or hematopoietic survival factors, or the direct effects of toxins and mediators of immune responses. Two common disorders associated with cell death are myocardial infarctions and stroke. In both disorders, cells within the central area of ischemia, which is produced in the event of acute loss of blood flow, appear to die rapidly as a result of necrosis. However, outside the central ischemic zone, cells die over a more protracted time period and morphologically appear to die by apoptosis.
Proteins containing a CARD domain are thought to be involved in various inflammatory disorders. For example, the CARD domain-containing protein caspase-1 promotes inflammation by converting certain proinflammatory cytokines from an inactive to an active form. Accordingly CARD-5 polypeptides, nucleic acids and modulators of CARD-5 expression or activity can be used to treat immune disorders. Such immune disorders include, but are not limited to, chronic inflammatory diseases and disorders, such as Crohn""s disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, Hashimoto""s thyroiditis and Grave""s disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g., leishmaniasis), certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy.
In addition to the aforementioned disorders CARD-5 polypeptides, nucleic acids, and modulators of CARD-5 expression or activity can be used to treat disorders of cell signaling and disorders of tissues in which CARD-5 is expressed.
The invention features a nucleic acid molecule which is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:1, SEQ ID NO:3, the nucleotide sequence of the cDNA insert of the plasmid deposited with the ATCC as Accession Number PTA-212 (xe2x80x9cthe cDNA of ATCC PTA-212xe2x80x9d), the nucleotide sequence of the cDNA insert of the plasmid deposited with the ATCC as Accession Number PTA-213 (the xe2x80x9ccDNA of ATCC PTA-213 xe2x80x9d), or a complement thereof.
The invention also features a nucleic acid molecule which includes a fragment of at least 150 (350, 400, 450, 500, 550, 600, 650, 700, and 761) nucleotides of the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the cDNA of ATCC PTA-212, or a complement thereof.
The invention also features a nucleic acid molecule which includes a fragment of at least 150 (350, 400, 450, 500, 550, 600, 650, 700, and 740) nucleotides of the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:6, the cDNA of ATCC PTA-213, or a complement thereof.
The invention features a nucleic acid molecule which includes a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:2, the amino acid sequence encoded by the cDNA of ATCC PTA-212, or the amino acid sequence encoded by the cDNA of ATCC PTA-213.
In another embodiment, a human CARD-5 nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:6 or the nucleotide sequence of the cDNA of ATCC PTA-213. In another embodiment, a murine CARD-5 nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.
Also within the invention is a nucleic acid molecule which encodes a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:2, the fragment including at least 15 (25, 30, 50, 100, 150, 300, 400 or 540, 600, 700, 800, 900) contiguous amino acids of SEQ ID NO:5, SEQ ID NO:2, the polypeptide encoded by the cDNA of ATCC Accession Number PTA-212, or the polypeptide encoded by the cDNA of ATCC Accession Number PTA-213.
The invention includes a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:2, or an amino acid sequence encoded by the cDNA of ATCC Accession Number PTA-212, or PTA-213, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:1, SEQ ID NO:3, the cDNA of ATCC PTA-212, or the cDNA of PTA-213 under stringent conditions.
Also within the invention are: an isolated CARD-5 protein having an amino acid sequence that is at least about 65%, preferably 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:5 and an isolated CARD-5 protein comprising an amino acid sequence that is at least about 90%, 95%, or 98% identical to SEQ ID NO:8 (CARD domain).
Also within the invention are an isolated CARD-5 protein having an amino acid sequence that is at least about 65%, preferably 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2 and an isolated CARD-5 protein comprising an amino acid sequence that is at least about 90%, 95%, or 98% identical to SEQ ID NO:7 (CARD domain).
Also within the invention are an isolated CARD-5 protein which is encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO:4 or the cDNA of ATCC PTA-213; an isolated CARD-5 protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 90% preferably 95%, or 98% identical to the CARD encoding portion of SEQ ID NO:4 (e.g., about nucleotides 383 to 596 of SEQ ID NO:4); and an isolated CARD-5 protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:4 or the non-coding strand of the cDNA of ATCC PTA-213.
Also within the invention are an isolated CARD-5 protein which is encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO:1; an isolated CARD-5 protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 90% preferably 95%, or 98% identical to the CARD encoding portion of SEQ ID NO:1 (e.g., about nucleotides 416 to 625 of SEQ ID NO:1); and an isolated CARD-5 protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1.
Another embodiment of the invention features CARD-5 nucleic acid molecules which specifically detect CARD-5 nucleic acid molecules, relative to nucleic acid molecules encoding other members of the CARD superfamily. For example, in another embodiment, a CARD-5 nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, or the cDNA of ATCC PTA-213, or a complement thereof. In another embodiment, the CARD-5 nucleic acid molecule is at least 300 (350, 400, 450, 500, 550, 585, 600, 650, 700, or 740) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:6, the cDNA of ATCC PTA-213, or a complement thereof. In another embodiment, an isolated CARD-5 nucleic acid molecule comprises nucleotides 383 to 596 of SEQ ID NO:4, encoding the CARD of CARD-5. In yet another embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a CARD-5 nucleic acid.
Another aspect of the invention provides a vector, e.g., a recombinant expression vector, comprising a CARD-5 nucleic acid molecule of the invention. In another embodiment the invention provides a host cell containing such a vector. The invention also provides a method for producing CARD-5 protein by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a CARD-5 protein is produced.
Another aspect of this invention features isolated or recombinant CARD-5 proteins and polypeptides. Preferred CARD-5 proteins and polypeptides possess at least one biological activity possessed by naturally occurring human CARD-5 e.g. (1) the ability to form protein:protein interactions with proteins in the apoptotic signaling pathway; (2) the ability to form CARDxe2x80x94CARD interactions with proteins in the apoptotic signaling pathway, e.g., caspase-1, CARD-12 or CARD-7; (3) the ability to bind a CARD-5 ligand; and (4) the ability to bind to an intracellular target. Other activities include: (1) modulation of cellular proliferation; (2) modulation of cellular differentiation; (3) modulation of cellular death; (4) modulation of the NF-xcexaB pathway; (5) modulation of proinflammatory cytokine activation; and (6) modulation of inflammation.
The CARD-5 proteins of the present invention, or biologically active portions thereof, can be operatively linked to a non-CARD-5 polypeptide (e.g., heterologous amino acid sequences) to form CARD-5 fusion proteins, respectively. The invention further features antibodies that specifically bind CARD-5 proteins, such as monoclonal or polyclonal antibodies. In addition, the CARD-5 protein or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
Preferred CARD-5 polypeptides includes at least 15, 25, 50, 60, 70, 80, 90, 100, 125, 150, or 190 contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:5. Preferred polypeptides comprise at least one domain present in CARD-5 (e.g., a CARD domain or a pyrin domain).
In another aspect, the present invention provides a method for detecting the presence of CARD-5 activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of CARD-5 activity such that the presence of CARD-5 activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating CARD-5 activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) CARD-5 activity or expression such that CARD-5 activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to CARD-5 protein. In other embodiments, the compound is one which interferes with the binding of the CARD domain of CARD-5 to another CARD-domain. In another embodiment, the agent modulates expression of CARD-5, by modulating transcription of a CARD-5 gene, splicing of a CARD-5 mRNA, or translation of a CARD-5 mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the CARD-5 mRNA or the CARD-5 gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant CARD-5 protein or nucleic acid expression or activity or related to CARD-5 expression or activity by administering an agent which is a CARD-5 modulator to the subject. In one embodiment, the CARD-5 modulator is a CARD-5 protein. In another embodiment the CARD-5 modulator is a CARD-5 nucleic acid molecule. In other embodiments, the CARD-5 modulator is a peptide, peptidomimetic, or other small molecule.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation of a gene encoding a CARD-5 protein; (ii) mis-regulation of a gene encoding a CARD-5 protein; (iii) aberrant RNA splicing; and (iv) aberrant post-translational modification of a CARD-5 protein, wherein a wild-type form of the gene encodes a protein with a CARD-5 activity.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of a CARD-5 protein. In general, such methods entail measuring a biological activity of a CARD-5 protein in the presence and absence of a test compound and identifying those compounds that alter the activity of the CARD-5 protein.
The invention also features assays for identifying compounds that reduce the interaction of CARD-5 with a CARD-5 ligand, e.g., caspase-1, CARD-7, CARD-12, or CARD-5. Isolated CARD domains of CARD-5, caspase-1, CARD-12 and/or CARD-7 can be used in these assays. The assays include measuring the binding of a CARD domain containing polypeptide to a polypeptide comprising the CARD domain of CARD-5 in the presence and absence of a test compound. A test compound is identified as a compound that reduces the binding of CARD-5 with a CARD-5 ligand if the binding of the polypeptides to each other is less in the presence of the test compound than in the absence of the compound. Additional compounds can be identified by measuring the binding of a polypeptide comprising the pyrin domain of CARD-5 to a polypeptide comprising a pyrin domain (e.g., NBS-1 or Pyrin-1).
The invention also features a method for identifying compounds that alter CARD-5-mediated apoptosis. The methods entail measuring apoptosis of cells that express a polypeptide comprising the pyrin domain of CARD-5 and cells that do not express (or express at a lower level) a polypeptide comprising the pyrin domain of CARD-5 in the presence and absence of a test compound. Test compounds that reduce the apoptosis of cells expressing the pyrin domain of CARD-5 relative to cells that do not express the pyrin domain of CARD-5 are candidate selective inhibitors of CARD-5 mediated apoptosis.
Reduction of CARD-5 activity or expression may play a role in breast cancer or other cancer. Accordingly, compounds that increase the activity or expression of CARD-5 may be useful for treatment of breast cancer or other cancers.
The invention also features methods for identifying a compound that modulates the expression of CARD-5 by measuring the expression of CARD-5 in the presence and absence of a compound.
Other features and advantages of the invention will be apparent from the following detailed description and claims.